Abstract: Congenital abnormalities and genetic diseases are a leading cause of infant mortality in the US1. While newborn screening (NBS) has dramatically reduced infant morbidity and mortality for some genetic disorders, these improvements have not had a significant impact in Neonatal Intensive Care Units (NICU) where 10 - 25% of all NICU admissions are the result of a genetic disease, with these infants staying in the hospital approximately 40% longer than those without genetic conditions. Due to the non-specific presentation of many of these genetic disorders, many infants do not receive a definitive diagnosis in a timely fashion, if at all. Large, comprehensive studies to determine the overall incidence of genetic disease in the neonatal population are lacking and have only recently been possible with the advent of next generation sequencing methodology such as exome and whole genome sequencing (WGS). Precise and rapid molecular diagnosis is needed to optimize clinical outcomes while reducing mortality and morbidity. In order to avoid the ethical, financial and technical aspects of exome and genome sequencing, we are introducing a rapid, targeted, next-generation sequencing (TNGS) panel that interrogates standard dried blood spots for genes matched to phenotypes affecting the neonatal population and has the potential to detect >98% of clinically relevant sequence variants for Mendelian inherited disorders with the highest morbidity and mortality. Here, we will conduct a multicenter prospective trial to examine the diagnostic efficacy, clinical utility and economic impact of a precision neonatal medicine approach through a public-private partnership among six leading CTSA sites and industry to further develop the TNGS methodology. We will characterize the time to diagnosis, time to initiation of appropriate treatment (or palliative care), and total costs in 400 high-risk neonates with signs/symptoms consistent with a genetic disorder, comparing standard diagnostic procedures to TNGS and WGS. This study aims to: 1) Assess the efficacy and the clinical utility of multiplexed (multi-gene) diagnostic tests (TNGS, WGS) for infants admitted to the NICU; 2) Examine the economic impact of clinical multiplexed sequencing in high-risk neonates compared with current standard of care diagnostic testing; and 3) Develop and evaluate the use of an electronic mechanism for accelerated results return (including any supporting documentation of existing treatments and open clinical trials). The overarching goal of this proposal is to examine the clinical utility and operational infrastructure of a neonatal gene panel in high-risk neonates in order to determine if it will provide a more timely diagnosis and better care at significantly lower cost than standard diagnostic care or WGS, establishing the foundation for a CTSA wide Neonatal Precision Medicine Program.